Polycyclic phthalazine compounds and their use

ABSTRACT

Polycyclic phthalazines, in particular 2-substituted maduraphthalazines (10,12,15,16-tetrahydroxy-8-methoxy-11-methyl-1,9,14-trioxo-1,2,6,7,9,14-hexahydronaphthaceno-1,2-g-phthalazines), of formula I and their use against gram-positive bacterial strains, particularly against multi-resistant staphylococci (MRSA) and against glycopeptide-resistant, for example vancomycin-resistant, enterococci. The polycyclic phthalazines are thus suitable for preparing anti-bacterially effective pharmaceutical compositions. In formula I, R 1  is carboxyalkyl or carboxyaryl, and R 2  and R 3  represent H or acyl (for example CO-alkyl or COO-alkyl), with R 1  being different from optionally substituted carboxyphenyl when R 3  represents C 1-8  alkanoyl. The invention also relates to salts, amides and esters of compounds of formula I.

BACKGROUND OF THE INVENTION

This invention relates to new polycyclic phthalazine derivatives,particularly 2-carboxyalkyl- and 2-carboxyaryl-substitutedmaduraphthalazines(10,12,15,16-tetrahydroxy-8-methoxy-11-methyl-1,9,14-trioxo-1,2,6,7,9,14-hexahydronaphthaceno-[1,2-g]-phthalazines)as well is salts, esters and amides thereof. They constitute substanceswhich exhibit a strong antibacterial effect against Staphylococci,particularly against gram-positive Staphylococci, and especially againstmulti-resistant Staphylococci (MRSA) and resistant Enterococci, and canbe used for combatting infectious diseases in humans and animals. Thesecompounds are suitable for the production of very differentpharmaceutical preparations and forms of application.

The compounds are the first representatives of a hitherto unknown ringsystem, namely of naphthaceno-[1,2-g]-phthalazine. They are derived frommadurahydroxylactone or maduranic acid, which can be obtained by abiotechnology route from Actinomadura rubra (W. Fleck, D. G. Strauss, J.Meyer, Z. Allg. Mikrobiol. 18, 368-398 (1978)). The structure ofmadurahydroxylactone (formula II) has been elucidated by Paulus andco-workers (E. F. Paulus, K. Dornberger, W. Werner, D. Fenske, ActaCryst. (1994) C50, 2064-2067). Madurahydroxylactone itself only exhibitsan unsatisfactory biological efficacy. Madurahydroxylactone forms partof the benzonaphthacenequinone class, from which benanomycins andpradimycins in particular have recently become known as antifungalsubstances of interest (T. Oki in "Recent Progress in AntifungalChemotherapy", Eds. H. Yamaguchi, G. S. Kobayachi, H. Takahashi, MarcelDekker, Inc., New York, Basle, Hong Kong, 1992, page 38).

SUMMARY OF THE INVENTION

The present invention serves to provide 2-substituted, particularly2-carboxyalkyl- and 2-carboxyaryl-substituted, Maduraphthalazines(10,12,15,16-tetrahydroxy-8-methoxy-11-methyl-1,9,14-trioxo-1,2,6,7,9,14-hexahydro-naphthaceno-[1,2-g]-phthalazines)and the corresponding salts, esters and amides, and also relates to theuse thereof. With these compounds, efforts have been made to increasethe range of antibacterial preparations, especially preparations whichare effective against grain-positive bacteria and which are effectiveagainst multi-resistant Staphylococci in particular. These new compoundsare advantageous for the achievement of this object because thecompounds or anti-infection preparations which were known hithertopossess serious disadvantages, such as insufficient effectivenessagainst super-resistant bacterial strains for example (W. Witte, C.Braulke, D. Heuck, C. Cuny; "Analysis of nosocomial outbreaks withmultiple- and methicillin-resistant Staphylococcus aureus (MRSA) inGermany: implications for hospital hygiene", Infection 22, (1994),Suppl. 2, 128-134; G. M. Eliopulos, "Increasing problems in the therapyof enterococcal infection", Eur. J. Clin. microbiol. Infect. Dis. 12(1993) 409-412.

This object is achieved according to the invention by the provision ofnew polycyclic phthalazine derivatives of general formula I ##STR1##wherein R¹ denotes carboxyalkyl or carboxyaryl and R² and R³,independently of each other, represent hydrogen or acyl, as well assalts, esters and amides of compounds of formula I, with the provisothat R¹ is different from a carboxyphenyl group, which is optionallysubstituted, if R³ denotes C₁₋₈ alkanoyl.

Within the scope of the present invention, the term "alkyl" (in compoundwords also, such as alkoxy or carboxyalkyl) denotes a C₁₋₁₀ alkyl whichmay be a straight-chain or branched-chain alkyl, preferably a C₁₋₄alkyl, whilst an aryl radical (including any one of the aryl radicalswhich are further cited below, or in compound words such as aralkyl) isto be understood to mean a substituted or unsubstituted aryl group,particularly a corresponding phenyl group, the substituents of which canbe OH, O-alkyl, O-aryl, a halogen, alkyl or aryl. Within the scope ofthe present invention, the term "acyl" primarily means a C₁₋₈ alkanoyl,C₁₋₈ alkoxycarbonyl or aroyl group, particularly a benzoyl or asubstituted benzoyl group (wherein the substituents are the same as fora substituted aryl), and optionally a carbamoyl group which issubstituted by one or two C₁₋₄ alkyl radicals.

Examples of corresponding R¹ radicals are those of formulae --(CR⁴R⁵)_(n) --COY or ##STR2## wherein R⁴ and R⁵ ═H, alkyl or aryl or asubstituted aryl, n=1-4, R⁶ ═H, alkyl, OH, alkoxy in the o-, m- orp-position or COY in the o-, m- or p-position, and Y═OR⁷, wherein R⁷ ═H,alkyl, aryl or a substituted aryl, an alkali metal ion, such as Na⁺ orK⁺ for example, or an ammonium ion (NH₄ ⁺ or a mono-, di- ortrialkylammonium ion or an N-methyl-D-glucammonium salt), or Y═NR⁸ R⁹,wherein R⁸ and/or R⁹ can be H, alkyl, aryl or a substituted aryl oraralkyl. If asymmetric C atoms are present, the present invention alsorelates to the corresponding D- and L-forms, enantiomers anddiastereoisomers, and also relates to racemates or mixtures ofenantiomers and diastereoisomers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the compounds of formula I which are particularly preferred, R¹represents carboxy-C₁₋₄ alkyl or C₁₋₄ alkoxycarbonyl-C₁₋₄ alkyl orrepresents carboxyphenyl, and R² and R³ represent hydrogen, wherein thecarboxy group exists in its free form or in salt form. The compounds offormula I and the salts thereof which are cited in the examples shouldbe emphasised.

The compounds according to the invention are prepared, for example, bythe reaction of madurahydroxylactone or maduranic acid(3,9,11,14,15-pentahydroxy-10-methyl-7-methoxy-1,8,13-trioxo-1,3,5,6,8,13-hexahydro-naphthaceno-[1,2-f]-benzoisofurane)of formula II ##STR3## with hydrazinoalkylcarboxylic acids orhydrazinophenylcarboxylic acids or esters and amides thereof, optionallyin the form of salts thereof. The reaction is conducted with the use ofsuitable solvents, e.g. glacial acetic acid. The temperature of reactionis usually the boiling temperature of the solvent, and the time ofreaction can range from one to a few hours.

Compounds of formula I which are prepared according to the invention canbe converted in the manner known in the art into other compounds offormula I. Compounds of formula I which can be obtained and whereinY═O-alkyl can thus be converted into the corresponding acids (formula Iwherein Y═OH) by alkaline saponification, e.g. with 2M caustic soda, andsubsequent acidification, e.g. with 2M hydrochloric acid. Alternatively,compounds of formula I which are obtained and in which R² or R³ ═H canbe converted into O-acyl derivatives, corresponding to formula I with R²or R³ ═H or acyl (e.g. --CO-alkyl or --COO-alkyl), by the acylation ofthe phenolic OH groups in a second step. This reaction step is conductedby employing customary methods for the acylation of phenolic OH groups,e.g. by means of an acid anhydride (e.g. acetic anhydride or propionicanhydride) or by means of esters of chloroformic acid (e.g. methylchloroformate) in alkaline solution, e.g. in 2M caustic soda or intetrahydrofuran/triethylamine. The reaction temperature can range from-20° C. to +20° C.

Compounds which are obtained in which Y═OH can be converted by customarymethods into the corresponding salts in which Y═OR⁷, wherein R⁷ can bean alkali metal ion (e.g. Na, K) or an ammonium ion (NH₄, a mono-, di-or trialkylammonium ion, e.g. a triethylammonium ion or anN-methyl-D-glucammonium ion). The synthesised compounds can be purifiedby means of customary methods (e.g. by recrystallisation or columnchromatography).

Compounds which are prepared according to the invention inhibit thegrowth of bacteria, particularly of gram-positive bacteria, especiallyof multi-resistant Staphylococci and of Enterococci. An aspect ofparticular importance is that the compounds are also highly effectiveagainst quinolone-resistant Staphylococci and are also highly effectiveagainst multiply-resistant strains (MRSA), and are even highly effectiveagainst glycopeptide-resistant strains, e.g. againstvancomycin-resistant hospital strains.

The minimum inhibiting concentration (MIC) of the compounds was tested,in a micro-broth dilution test according to DIN 58 940 (Part 8), againstthe following bacterial strains: Staphylococcus aureus, strains 8325-4(a sensitive Staphylococcus aureus strain which is representative of thespecies), NCTC 6571 (a sensitive international control strain), 108/83(an "old" MRSA without quinolone-resistance), 134/94 (a "new" MRSA, withquinolone-resistance), Staphylococcus epidermidis CCM 2124 (a sensitivecontrol strain), Enterococcus faecium 70/90 (a vancomycin-resistantstrain) and 64/3 (a sensitive strain). The known substances vancomycin,teicoplanin and ciprofloxacin were used for comparison in the tests,even though they are structurally different.

The results of the antibacterial testing are compared in the Table. Itfollows from the results that the substances which were preparedaccording to the invention exhibit inhibiting effects against somebacterial strains which are significantly superior to those of thecomparison substances, and are capable of successfully overcomingbacterial resistance.

On account of their antibacterial properties, compounds of generalformula I are suitable for use as drugs for bacterial infections,particularly infection by multi-resistant Staphylococci. For diseasessuch as these, compounds of formula I can be used either on their own orwith physiologically compatible adjuvant substances or supportsubstances, wherein all customary pharmacological forms of applicationand physiologically compatible dosages are possible in principle.Application is effected orally or parenterally for example, e.g.intravenously.

EXAMPLES Example 1

Substance 1

2-(4-carboxyphenyl)-maduraphthalazine(2-(4-carboxyphenyl-10,12,15,16-tetrahydroxy-8-methoxy-11-methyl-1,9,14-trioxo-1,2,6,7,9,14-hexahydro-naphthaceno-[1,2g]-phthalazine), formula I wherein R¹ ═4-carboxyphenyl, R², R³ ═H, C₃₃H₂₂ N₂ O₁₀ (606.54).

A mixture of 100 mg (0.2 mmoles) madurahydroxylactone (80 percent) and50 mg 4-hydrazino-benzoic acid (0.2 mmoles) was boiled under reflux in10 ml glacial acetic acid for 4 hours. The red crystals obtained werewashed with ether.

The substance was purified twice via preparative DC (Merck ready-to-use1 mm Si 60 plates, mobile phase CH₂ Cl₂ /CH₃ OH (9:1)), dissolved inTHF, filtered and precipitated with petroleum ether. Yield 78 mg (63%theoretical), m.p.>350° C., purity as determined by HPLC (Eurospher,acetonitrile/water (3:2)): 94,7%, t=9.4 minutes, DC (Merck Alufolie Si60), mobile phase CH₂ Cl₂ /CH₃ OH (9:1)), R_(f) =0.77,

MS FAB (3NBA) [M+H]⁺ : mass number found=607.1, ¹ H NMR (DMSO-D₆): d(ppm)=14.1, 13.6, 12.98, 11.2 (4H, s, 10-,12-,15-,16-OH) 8.6 (1H, s,4-CH) 7.3 and 7.5 (2×1H, s, 5-CH and 13-CH), 7.8, 7.83, 8.08, 8.12 (4H,2-phenylene), 3.85 (3H, s, 8-OCH) 2.1 (3H, s, 11-CH₃).

Example 2

Substance 2

2-ethoxycarbonylmethyl-maduraphthalazine

(10,12,15,16-tetrahydroxy-8-methoxy-2-ethoxycarbonyl-methyl-11-methyl-1,9,14-trioxo-1,2,6,7,9,14-hexahydro-naphthaceno-[1,2-g]-phthalazine),formula I wherein R¹ =CH₂ COOC₂ H₅, R², R³ ═H, C₃₀ H₂₄ N₂ O₁₀ (572.53).

A mixture of 1 g maduranic acid (97 percent) (2 mmoles) and 400 mg ethylhydrazinoacetate hydrochloride (2.5 mmoles) was boiled in 20 ml glacialacetic acid with stirring for 2 hours. After filtration under suction,the product was well washed with water and glacial acetic acid and wasrecrystallised from glacial acetic acid. Light red crystals, m.p.324-325° C. (decomposition). Yield: 622 mg (54% theoretical). Purity asdetermined by HPLC (Nucleosil RP18, acetonitrile/water (3:2)): 98.14%,t=10.45 min, DC (Merck Alufolie Si 60): butyl acetate/glacial aceticacid (4:1): R_(f) =0.87, MS FAB (3NBA) [M+H]⁺ : mass number found=573.1,¹ H NMR (DMSO-D₆) d(ppm): 14.07, 13.56, 12.77, 11.18 (4×H, s, 10, 12, 15and 16-OH) 8.50 (1H, s, 4-CH), 7.40 and 7.28 (2H, s, 5-CH and 13-CH),4.97 (2H, s, NCH₂) 4.15-4.25 (2H, q, CH₂ of ethyl), 3.81 (3H, s, 8-OCH₃)2.08 (3H, s, 10-CH₃), 1.20-1.268 (3H, t, CH₃ of ethyl).

Example 3

Substance 3

2-carboxymethyl-maduraphthalazine

(10,12,15,16-tetrahydroxy-8-methoxy-2-carboxynmethyl-11-methyl-1,9,14-trioxo-1,2,6,7,9,14-hexahydro-naphthaceno-[1,2-g]-phthalazine),formula I wherein R¹ ═CH₂ COOH, R², R³ ═H, C₂₈ H₂₀ N₂ O₁₀ (544.47).

570 mg (1 mmole) 2-carbethoxymethyl-maduraphthalazine (substance 2) wereallowed to stand for 1 day in 35 ml of 2M caustic soda solution. Theproduct was subsequently acidified with 2M hydrochloric acid. The redcrystals obtained were purified by recrystallisation or by boiling inglacial acetic acid or by dissolution in tetrahydrofuran andprecipitation with petroleum ether m.p. 234-236° C. (decomposition),yield: 322 mg (59% theoretical). Purity as determined by HPLC (NucleosilRP18, acetonitrile/water (3:2), 99.8% t=0.87 minutes, DC (Merck AlufolieSi 60): butyl acetate/glacial acetic acid (4:1): R_(f) =0.31, MS FAB(3NBA) [M+H]⁺ : mass number found=545.0. ¹ H NMR (CDCl₃): d (ppm): 14.0,13.5, 12.8, 11.2 (4H, s, 10,12,15 and 16-OH) 8.42 (1H, s, 4-CH), 7.2 and7.3 (2H, s, 5-CH and 13-CH), 3.9 (3H, s, 7-OCH₃) 2.9 and 2.5 (2×2H, s,6- and 7-CH₂), 2.1 (3H, s, 10-CH₃), ¹³ C NMR (CDCl₃) 28 C atoms; d(ppm): 187.66 and 185.54 (quinone group), 169.0 (2-COOH), 61.04 (OCH₃),52.30 (NCH₂), 29.21 and 22.21 (6- and 7-CH₂), 8.17 (11-CH₃). ¹³ C NMRDEPT 135: d (ppm): 139.44 (CH═N) 114.19, 106.06 (2×ArH), 60.79 (8-OCH₃),52.04 (NCH₂), 29.05, 21.96 (2×CH₂), 7.92 (11-CH₃).

A water-soluble trialkylammonium salt, for example a triethylammoniumsalt or an N-methyl-D-glucammonium salt, could be obtained fromsubstance 3 by the addition of an amine, for example triethylamine orN-methyl-D-glucamine, in a suitable solvent, for exampletetrahydrofuran.

Example 4

Substance 4

2-(D,L-a-carboxypropyl)-maduraphthalazine(10,12,15,16-tetrahydroxy-8-methoxy-2-(D,L-a-carboxypropyl-11-methyl-1,9,14-trioxo-1,2,6,7,9,14-hexahydro-naphthaceno-[1,2-g]-phthalazine),formula I wherein R¹ -50 D,L-a-carboxy-propyl, R², R³ ═H, C₃₀ H₂₄ N₂ O₁₀(572.15).

A mixture of 2 g (4.1 mmoles) madurahydroxylactone and 0.52 g (4.4mmoles) D,L-a-hydrazinobutyric acid was boiled in 50 ml glacial aceticacid for 2 hours under reflux. After standing overnight, the dark redcrystals were filtered off under suction, washed with ether andrecrystallised from glacial acetic acid. Yield 1.8 g (76% theoretical),m.p. 225-230° C. (decomposition), purity as determined by HPLC (RP18Eurospher 100 C7, acetonitrile/water (3:2)+0.05 percent trifluoroaceticacid): 99.6%, t=13.3 min, DC (Merck Alufolie Si 60): butylacetate/glacial acetic acid (4:1): R_(f) =0.79, MS FAB (3NBA) [M+H]⁺ :mass number found=573.4, ¹ H NMR (DMSO-D6) d (ppm): 14.1, 13.6, 12.9,11.3 (4H, s, 10, 12, 15 and 16-OH) 8.5 (1H, s, 4-CH), 7.2 and 7.4 (2H,s, 5-CH and 13-CH), 5.3 (1H, d, NCH), 3.9 (3H, s, 7-OCH₃), 2.0 (3H, s,10CH₃), 0.9 (3H, s, CH₃ aliph.). ¹³ C NMR (DMSO-D6) Dept 135: d (ppm):29.48, 22.43, 22.22 (3×CH₂).

The abbreviations used in the examples have the following meanings

DC - thin layer chromatography

THF - tetrahydrofuran

HPLC - high performance liquid chromatography

min - minutes

m.p. - melting point

MS - mass spectrometry

NMR - nuclear magnetic resonance

                                      TABLE                                       __________________________________________________________________________    Effectiveness of maduraphthalazine derivatives against various                gram-positve bacterial strains                                                MIC (mg/ml)           Staphylococcus                                                                           Enterococcus                                 Staphylococcus aureus                                                                           NCTC                                                                              epidermidis                                                                              faecium                                      Substance                                                                           8325-4.sup.a)                                                                     108/83.sup.b)                                                                     134/94.sup.c)                                                                     6571                                                                              CCM 2124                                                                             70/90                                                                             64/3                                         __________________________________________________________________________    1.    0.5 0.25                                                                              0.5 <0.125                                                                            <0.125 0.5 0.5                                          3.    0.063                                                                             0.063                                                                             0.25                                                                              <0.125                                                                            <0.125 2   0.5                                          4.    0.063                                                                             0.032                                                                             0.016                                                           vancomycin                                                                          0.5 0.5 0.5     0.5    1024                                                                              <0.25                                        teicoplanin                                                                         0.5 0.5 0.5     0.5    256 <0.25                                        ciprofloxacin                                                                       0.25                                                                              0.25                                                                              16.0    0.25   1   <0.25                                        __________________________________________________________________________     .sup.a) a sensitive Staphylococcus aureus strain, representative of the       species                                                                       .sup.b) "old" MRSA (without quinoloneresistance)                              .sup.c) "new" MRSA (with quinolineresistance)                            

What is claimed is:
 1. A polycyclic phthalazine compound correspondingto the formula: ##STR4## wherein R¹ represents a carboxyalkyl orcarboxyaryl group;R² and R³ independently represent hydrogen or an acylgroup, or a salt, ester or amide thereof; with the proviso that R¹ isother than a carboxyphenyl or substituted carboxyphenyl group when R³represents a C₁₋₈ alkanoyl group.
 2. A compound according to claim 1,wherein R¹ represents a group corresponding to the formula --(CR⁴R⁵)_(n) --COY or ##STR5## wherein R⁴ and R⁵ represent H, alkyl, aryl orsubstituted aryl;n is an integer from 1 to 4; R⁶ represents H, alkyl, OHor alkoxy, and Y represents OR⁷ wherein R⁷ represents H, alkyl, aryl,substituted aryl, an alkali metal ion or an ammonium ion, or Yrepresents NR⁸ R⁹, wherein R⁸ and R⁹ independently represent H, alkyl,aryl, or substituted aryl or aralkyl.
 3. A compound according to claim1, whereinR¹ represents carboxy-C₁₋₄ alkyl, C₁₋₄ alkoxycarbonyl-C₁₋₄alkyl or carboxyphenyl, and R² and R³ represent hydrogen.
 4. A compoundaccording to claim 3, in salt form.
 5. A compound according to claim 3,in free acid form.
 6. A compound according to claim 1, in salt form. 7.A compound according to claim 1, in free acid form.
 8. A compoundaccording to claim 2, wherein R⁷ represents a trialkylammonium ion or aN-methyl-D-glucammonium ion.
 9. A compound according to claim 1, whereinsaid compound is 2-carboxymethylmaduraphthalazine or a salt thereof. 10.A pharmaceutical composition comprising an effective antibacterialamount of a compound according to claim 1, and at least onepharmaceutical carrier or adjuvant.
 11. A method of preparing apharmaceutical composition comprising admixing an effectiveantibacterial amount of a compound according to claim 1, with at leastone pharmaceutical carrier or adjuvant.
 12. A method of treating abacterial infection in a patient, said method comprising administeringto said patient an effective antibacterial amount of a compoundaccording to claim 1.